Viral RNA genomes show remarkable functional versatility. Evolutionary constraints have led RNA viruses to develop high information densities in every aspect of viral structure; as an example, viruses utilize frameshifting to expand their proteomes while not expanding their genomes, and this process is regulated by RNA structural elements. The secondary structure of an entire HIV-1 genome has recently been determined, revealing numerous ways in which genome replication, recombination, and even protein structure are encoded in RNA structures. These discoveries at the level of RNA secondary structure are increasing our understanding of HIV-1 replication and will likely reveal principles common to other RNA- based pathogens and to RNA metabolism, in general. The next frontier is to understand the roles of tertiary structure in intact RNA genomes. The goal of this research is to experimentally locate sites of higher-order tertiary structure using authentic HIV-1 genomic RNA. I hypothesize that these sites will be relatively rare, but will occur in regions of particularly high functional significance, consistent with their probable regulatory roles. I will use two approaches to locate tertiary structure: hydroxyl radical probing, and differential SHAPE reactivity. Hydroxyl radical probing is a well-established technique in which protection from cleavage reveals areas where bulk solvent cannot contact the RNA backbone. These areas are strongly correlated with higher-order RNA structure. Differential SHAPE reactivity takes advantage of previous discoveries in the Weeks lab that established that highly constrained RNA nucleotides in unusual conformations tend to show exceptionally slow local nucleotide dynamics. A large fraction of these nucleotides form the relatively rare ribose C2'-endo sugar conformation, and can be selectively detected as they react with slow, but not fast reacting, SHAPE reagents. These nucleotides occur disproportionately often in regions of functional importance and can dramatically alter the folding profile, even of large RNAs. Identification of these constrained residues pinpoints nucleotides with important structural roles, providing complementary and, in some ways, more specific information than hydroxyl radical probing. This work is innovative, as both of these assays will be performed on an entire authentic ex virio HIV-1 genome and will be analyzed using high-throughput technologies developed in the Weeks lab. The expected outcome of this research is a catalogue of regions of tertiary structure in the HIV-1 genome and information that can be used to develop novel hypothesis regarding the contributions of higher-order RNA structure in retroviral replication. Pursuit of this research proposal will also provide training in bioinformatics, virology, RNA biology, and high- throughput structure analysis and will, over the long term, facilitate identification of novel targets for antiviral interventions.